Mammals require molecular oxygen (O.sub.2) for essential metabolic processes including oxidative phosphorylation in which O.sub.2 serves as electron acceptor during ATP formation. Systemic, local, and intracellular homeostatic responses elicited by hypoxia (the state in which O.sub.2 demand exceeds supply) include erythropoiesis by individuals who are anemic or at high altitude (Jelkmann (1992) Physiol. Rev. 72:449-489), neovascularization in ischemic myocardium (White et al. (1992) Circ. Res. 71:1490-1500), and glycolysis in cells cultured at reduced O.sub.2 tension (Wolfle et al. (1983) Eur. J. Biochem. 135:405-412). These adaptive responses either increase O.sub.2 delivery or activate alternate metabolic pathways that do not require O.sub.2. Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza (1994) Hematol. Oncol. Clinics N. Amer. 8:863-884), vascular endothelial growth factor (Shweiki et al. (1992) Nature 359:843-845; Banai et al. (1994) Cardiovasc. Res. 28:1176-1179; Goldberg & Schneider (1994) J. Biol. Chem. 269:4355-4359), and glycolytic enzymes (Firth et al. (1994) Proc. Natl. Acad. Sci. USA 91:6496-6500; Semenza et al. (1994) J. Biol. Chem. 269:23757-23763).
The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and thus blood O.sub.2 -carrying capacity (Jelkmann (1992) supra; Semenza (1994) supra). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 31'-flanking region and a trans-acting factor that binds to the enhancer, hypoxia-inducible factor 1 (HIF-1), fulfilled criteria for a physiological regulator of EPO transcription: inducers of EPO expression (1% O.sub.2, cobalt chloride [CoCl.sub.2 ], and desferrioxamine [DFX]) also induced HIF-1 DNA binding activity with similar kinetics; inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity; and mutations in the EPO 3'-flanking region that eliminated HIF-1 binding also eliminated enhancer function (Semenza (1994) supra). These results also support the hypothesis that O.sub.2 tension is sensed by a hemoprotein (Goldberg et al. (1988) Science 242:1412-1415) and that a signal transduction pathway requiring ongoing transcription, translation, and protein phosphorylation participates in the induction of HIF-1 DNA-binding activity and EPO transcription in hypoxic cells (Semenza (1994) supra).
EPO expression is cell type specific, but induction of HIF-1 activity by 1% O.sub.2, CoCl.sub.2, or DFX was detected in many mammalian cell lines (Wang & Semenza (1993a) Proc. Natl. Acad. Sci. USA 90:4304-4308), and the EPO enhancer directed hypoxia-inducible transcription of reporter genes transfected into non-EPO-producing cells (Wang & Semenza (1993a) supra; Maxwell et al. (1993) Proc. Natl. Acad. Sci. USA 90:2423-2427). RNAs encoding several glycolytic enzymes were induced by 1% O.sub.2, CoCl.sub.2, or DFX in EPO-producing Hep3B or non-producing HeLa cells whereas cycloheximide blocked their induction and glycolytic gene sequences containing HIF-1 binding sites mediated hypoxia-inducible transcription in transfection assays (Firth et al. (1994) supra; Semenza et al. (1994) supra). These experiments support the role of HIF-1 in activating homeostatic responses to hypoxia.